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Laboratory of Molecular Microbiology and Biotechnology, Department of Biology, Faculty of Sciences, University of Chile, Santiago 1, Casilla 653, Santiago, Chile
Correspondence
Carlos A. Jerez
cjerez{at}uchile.cl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). PolyP is a linear polymer of hundreds of orthophosphate residues linked by phosphoanhydride bonds that has a variety of physiological functions, such as serving as a reservoir of phosphate, substitution for ATP in kinase reactions, serving as a chelator of metals, and adaptation to different stress conditions (Kornberg et al., 1999). The main enzyme involved in the biosynthesis of polyP is polyphosphate kinase (PPK), which catalyses the reversible conversion of the terminal phosphate of ATP into polyP (Akiyama et al., 1992). An exopolyphosphatase (PPX), on the other hand, is known to hydrolyse polyP, liberating inorganic phosphate (Pi) (Akiyama et al., 1993). These enzymes have been purified from Escherichia coli and their genes have been identified in several bacteria (Kornberg et al., 1999; Alvarez & Jerez, 2004).
One of the proposed mechanisms for metal tolerance is the sequestration of metal cations by polyP (Kornberg et al., 1999). Many heavy metal resistance systems involve either an active efflux or a detoxification of metal ions by different transformations (Silver & Phung, 1996). For copper, these include intracellular complexation, decreased accumulation, extracellular complexation or sequestration in the periplasm (Rouch et al., 1989; Harwood & Gordon, 1994). It has also been proposed that the hydrolysis of polyP detoxifies the metals (Aiking et al., 1984; Keasling, 1997). Van Veen (1997) has shown that the inorganic phosphate transport system (Pit) in Escherichia coli and Acinetobacter johnsonii can reversibly transport metal phosphates. Keasling & Hupf (1996), using genetically engineered strains of E. coli, obtained results indicating that not only a large quantity of intracellular polyP is important for tolerance to heavy metals but also the ability to synthesize and degrade polyP. Based on these results and those mentioned above, Keasling (1997) proposed a model in which the intracellular cation concentration in bacteria would regulate the activity of PPX, which would in turn degrade polyP, and the Pi generated accompanied by cation transport would be removed from the cell through the Pit system. We have recently found that Acidithiobacillus ferrooxidans, a micro-organism that can tolerate very high concentrations of heavy metals in its normal environment, normally accumulates high amounts of polyP granules and that the levels of intracellular polyP are greatly decreased when the bacterium is grown in or shifted to 100 mM Cu2+. In the presence of this metal, PPX activity and Pi efflux greatly increased, supporting a model for metal tolerance mediated through polyP (Alvarez & Jerez, 2004).
Related to archaeal copper resistance mechanisms, some metal efflux pumps have been identified in the genomes of archaea (Pedone et al., 2004). The P-type CPX-ATPases are responsible for the transport of heavy metal ions in all kinds of organisms. One of the two CPX-ATPases of Sulfolobus solfataricus has recently been isolated and characterized (Deigweiher et al., 2004). In Ferroplasma acidarmanus the Fer1 copper resistance (cop) loci (Ettema et al., 2003), which include genes encoding a putative transcriptional regulator (copY), a putative metal-binding chaperone (copZ) and a putative copper-transporting P-type ATPase (copB) have been described (Baker-Austin et al., 2005). By transcription analyses the authors demonstrated that copZ and copB are co-transcribed, and that the transcript levels increase significantly in response to exposure to high levels of Cu2+ ions, suggesting that the transport system is operating for copper efflux.
In archaea, polyP has been reported only in Methanosarcinae (Scherer & Bochem, 1983) and in Sulfolobus acidocaldarius (Skórko et al., 1989). However, nothing is known about the possible role of polyP in archaea. A partially purified PPK was reported in S. acidocaldarius (Skórko et al., 1989). However, we demonstrated later that this protein was rather a glycogen synthase (Cardona et al., 2001). Despite the fact that no bacterial-type PPKs have been found in the majority of the available archaeal genomic sequences, we recently reported the isolation of a PPX enzyme from Sulfolobus solfataricus (Cardona et al., 2002). In the present work, we have studied the effect of different metals in several Sulfolobus species and have found that the presence of these cations decreased polyP levels and increased both PPX activity and phosphate efflux, clearly supporting the proposed metal tolerance model.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. S. solfataricus DSM 1617 was grown at 70 °C in medium 182 (Deutsche Sammlung von Mikroorganismen und Zellkulturen) with 0·1 % (w/v) yeast extract and 0·1 % (w/v) Casamino acids. To study copper or cadmium tolerance of S. metallicus and S. solfataricus, the micro-organisms were grown in their respective media, except that different concentrations of CuSO4 (5–200 mM) or CdSO4 (0·01–3 mM) were present initially, as indicated in the corresponding experiments. For shift experiments, S. metallicus cells were grown in the absence of copper to the early stationary phase and after removing the medium from the cells by centrifugation, they were then shifted to a new medium containing 10, 20 or 100 mM CuSO4. As a control, a shift of cells to 100 mM (NH4)2SO4 was used. Aliquots were taken at different times and polyphosphate levels and PPX activity were determined. Growth was monitored by measuring unstained cell numbers by means of a Petroff–Hausser chamber under a phase-contrast microscope.
Electron microscopy.
Unstained cells from S. metallicus or S. solfataricus were routinely examined for the presence of electron-dense bodies by transmission electron microscopy (Gonzalez & Jensen, 1998). Early stationary-phase cells from different growth conditions were suspended in distilled water and then placed onto carbon-coated nickel grids. After allowing the micro-organisms to settle on the grid, the excess liquid was drained off with filter paper, and the preparations were air-dried. For analysis, a transmission electron microscope (Philips Tecnai 12) operating at 80 kV was used.
Electron energy loss spectroscopy (EELS).
EELS analysis (Lünsdorf et al., 2000) was performed with a Zeiss CEM 902 integrated energy-filtered transmission electron microscope. The microscope was operated in the electron spectroscopic imaging (ESI) mode for element mapping, and parallel EELS performed for spectrum registration with the aid of ESI-Vision software (Soft Imagining System). Aperture settings described by Lünsdorf et al. (2000) were used. Commercial hydroxyapatite was used as the internal phosphate standard (Chávez et al., 2004).
PolyP quantification.
Purified recombinant His6-PPK was prepared by using E. coli strain NR 100 as described previously (Ahn & Kornberg, 1990; Kumble et al., 1996), and this preparation was used in the polyP assay described below. The protein concentration was determined by the method of Bradford (Coomassie Plus protein assay reagent, Pierce).
PolyP was quantified by using a two-step conversion of polyP into ATP by PPK and quantification of ATP by using luciferase to generate light (Ault-Riché et al., 1998). First, polyP was extracted by using Glassmilk, from the different cells grown in the absence or in the presence of copper once they reached the early stationary phase. The polyP extracted was assayed by using the reverse reaction of E. coli PPK in ADP excess. Finally, the ATP generated was determined by using the luciferase (Boehringer Mannheim) reaction, and the luminescence was measured with a luminometer (BioScan Lumi/96). Concentration of polyP was expressed in terms of Pi residues.
In vitro preparation of [33P]polyP750 as a substrate for PPX.
Radioactively labelled polyP with a chain length of 750 residues was prepared as described by Ault-Riché et al. (1998), with the modifications of Cardona et al. (2001) and Alvarez & Jerez (2004). The identity and purity of [33P]polyP750 were determined by its susceptibility to hydrolysis by ScPPX1 as described previously (Ault-Riché et al., 1998; Cardona et al., 2001).
Preparation of cell-free extracts from S. metallicus.
Cells from 800 ml of a control culture grown to 108 cells ml–1, or cultures shifted to different CuSO4 concentrations or to 100 mM (NH4)2SO4, were harvested by centrifugation (7700 g for 15 min). The pellets were washed with M88 medium to remove the sulfur. Then the cells were resuspended in 50 mM Tris/acetate (pH 7·0)-10 % sucrose buffer (20 µl per mg wet weight), frozen, and sonicated eight times for 30 s each time. The lysate was centrifuged (4300 g for 5 min) to eliminate cellular debris, and the supernatant was used to measure PPX activity.
Assay for PPX activity and TLC analysis of the reaction products.
PPX activity was determined as described by Cardona et al. (2002), with the following modifications. The 50 µl reaction mixture contained 50 mM Tris/acetate (pH 7·0), 1 mM MnCl2, 100 mM KCl, 50 µg extract protein and 250 µM [33P]polyP750. After incubation of the mixture for 60 min at 65 °C, the reactions were stopped; 4 µl was taken from each reaction mixture and loaded on polyethyleneimine-cellulose plates (Merck). For TLC, samples of 4 µl were developed in 0·75 M KH2PO4 (pH 3·5). Radioactive spots were visualized and quantified by using a Phosphorimager (Molecular Imager FX Systems, Bio-Rad). One unit of enzyme was defined as the amount releasing 1 pmol phosphate from polyP min–1.
In vivo labelling of S. metallicus with 32Pi.
Cells were grown in medium 88 supplemented with 0·05 % (w/v) sulfur and 0·02 % (w/v) yeast extract to mid-exponential phase in Pi-sufficient conditions (2 mM Pi). Cells were collected by centrifugation and resuspended at a higher cell density (1010 cells ml–1) in a medium with a lower Pi concentration (0·2 mM Pi). To label the cells, 32H3PO4 [100 µCi ml–1 (3·7 MBq ml–1)] was added and the micro-organisms were further incubated for 20 h, after which the radioactively labelled cells were harvested by centrifugation.
Pi efflux measurements.
The 32Pi-labelled cells were exhaustively washed (five times) by resuspension and centrifugation with fresh medium 88 containing sufficient Pi (2 mM) to eliminate the non-incorporated radioactive label and finally were resuspended in the same medium to a cell density of 108 cells ml–1, in the presence or absence of CuSO4. To determine the amount of 32Pi released to the medium, samples (1·5 ml) were taken periodically and the supernatants obtained by their centrifugation at 12 000 g for 10 min were measured by scintillation counting and analysed by TLC on polyethylenimine-cellulose with 32Pi as a standard (Keasling et al., 1993).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). To detect the presence of these granules in members of the genus Sulfolobus, we analysed unstained cells of the micro-organisms by electron microscopy (Fig. 1a, b). When S. solfataricus (Fig. 1a) or S. acidocaldarius (not shown) was examined, very faint and small granules, if any, were present. In contrast, two or three large electron-dense granules per cell were seen in S. metallicus (Fig. 1b). To obtain detailed information about the polyphosphate-like inclusions, ultrastructural determinations were made by using scanning electron microscopy coupled to EELS analysis with an integrated energy-filtered transmission electron microscope. Fig. 1(c) shows the electron energy loss spectra obtained from cell areas containing granules in S. metallicus and in S. acidocaldarius compared with that for hydroxyapatite as a phosphate reference after background subtraction and spectrum filtering. The ionization energy onset of the PL2,3 edge is at 135 eV for all three spectra, followed by characteristic energy-loss near edge structures (ELNES) features in the 50 eV higher energy loss region (indicated by the bar in Fig. 1c). All three main maxima, i.e. I, II and III, were present in the spectra obtained, although the intensities in S. acidocaldarius were very small due to its low content of polyP granules, which was very similar to that of S. solfataricus (not shown). Thus, by using electron microscopic microanalyses we demonstrated that the electron-dense granules accumulated by S. metallicus were mainly composed of phosphate and most likely corresponded to polyP.
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). Using cell-free extracts obtained for each growth time point, polyP was enzymically quantified. All three micro-organisms synthesized increasing amounts of polyP as growth proceeded, levelling off when the stationary phase was reached.
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). The levels of 180 nmol polyP (mg protein)–1 (230 mM) reached at the stationary phase of growth (Fig. 2a
) are roughly 10-fold greater than those seen in S. solfataricus [20 nmol polyP (mg protein)–1 or 1 mM] (Fig. 2b) and S. acidocaldarius [10 nmol polyP (mg protein)–1 or 0·5 mM] (Fig. 2c). The amounts of polyP accumulated by S. metallicus are even higher than those reported (150 nmol mg–1) for another micro-organism accumulating polyP granules – Vibrio cholerae during the exponential phase of growth with a rich medium (Ogawa et al., 2000) – and are closer to the 400 nmol (mg protein)–1 accumulated by A. ferrooxidans (Alvarez & Jerez, 2004). It is possible that the polyP accumulations observed in these crenarchaea contribute not only to their energy supply but also to their enhanced survival and stress resistance in the presence of metals, as suggested for bacteria (Keasling, 1997; Alvarez & Jerez, 2004).
Effect of CuSO4 and CdSO4 in the growth of S. metallicus and S. solfataricus
The S. metallicus cells utilized in these experiments were previously adapted to grow at different concentrations of metals. There was a small decrease in the growth rates and the curves reached the plateaus at slightly lower cell densities when the micro-organisms were grown in the presence of increasing Cu2+ concentrations compared with the control culture in the absence of added Cu2+ (Fig. 3a). However, at 200 mM Cu2+, there was a more severe effect of copper on growth, with an approximately 30 % decrease in cell numbers. On the other hand, S. solfataricus was not able to grow in any of the copper concentrations tested (Fig. 3b). These results are in agreement with previous reports in which S. solfataricus was able to grow only in the presence of 0·1–1·0 mM copper, being completely inhibited by growth in the presence of 10 mM copper (Miller et al., 1992). This behaviour was similar to that seen for S. acidocaldarius, which also did not grow in the presence of 10 mM Cu2+ (Miller et al., 1992). These results clearly contrast with the very high tolerance of S. metallicus to concentrations of copper sulphate as high as 100 and 200 mM (Fig. 3a). S. metallicus could grow in the presence of CdSO4 concentrations up to 1 mM without a noticeable effect in the cell numbers obtained at the stationary phase. At higher concentrations (2–3 mM) there was an extended lag period and the culture reached significantly lower cell numbers (results not shown). On the other hand, S. solfataricus growth was seriously affected by CdSO4 concentrations as low as 10 µM (results not shown).
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a). This behaviour correlated exactly with the disappearence of the polyP granules observed when cells were subjected to those high copper levels (Fig. 4b, c, d). In the absence of copper, 96 % of the cells contained two or three large polyP granules per cell (Fig. 4b), whereas at 200 mM copper sulphate the granules were very small, with only 46 % of the cells containing only one of these aggregates (Fig. 4d). These results strongly indicate a possible relationship between the polyP levels and the adaptation of S. metallicus to grow in the presence of Cu2+.
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). Copper greatly stimulated PPX activity at very low concentrations. The same maximal PPX activity was reached with both copper and manganese. However, the metal concentration requirement for this was 10 µM for copper (Fig. 5) and 500 µM for manganese (results not shown). At concentrations of copper higher than 10 µM there was an inhibition of the activity, whereas manganese continued to stimulate PPX. Other metals such as cadmium and zinc also stimulated PPX activity in the micromolar range although to a lower extent (results not shown). These results suggest a stimulatory effect of copper and other metal ions on PPX activity and polyP hydrolysis.
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a). No significant effect on PPX activity or polyP levels was seen when the cells were shifted to 100 mM (NH4)2SO4 (results not shown). The increase in PPX activity corresponded in time with the decrease in polyP levels seen in Fig. 6(b), strongly suggesting that the decrease in polyP levels observed when cells were shifted to the presence of copper was due to an increase of this PPX activity.
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).
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) and the Pi released (Fig. 7) were observed. A similar situation has been found in Acinetobacter johnsonii subjected to conditions (anaerobiosis) in which 65 % of the polyP is degraded, since intracellular Pi accumulated up to 150 mM in the course of polyP degradation, while the external Pi concentration remained below 11 mM. The amount of 32Pi released to the medium was the highest in cells exposed to 200 mM Cu2+. By analysing the radioactivity released to the medium by TLC on polyethyleneimine-cellulose we found that it consisted mainly of Pi (not shown). No other radioactively labelled cellular metabolites appeared on the TLC, indicating that the label released to the medium was not the product of cellular lysis during the treatment with copper. These results strongly suggest that at least part of the Pi generated from polyP hydrolysis in the presence of Cu2+ ions was transported out of the cells. Formation and transport of metal–phosphate complexes have been demonstrated in cells or isolated membrane vesicles from bacteria by determination of changes in the membrane potentials (van Veen et al., 1994). This type of system is not currently available in S. metallicus. Our attempts to label S. metallicus cells with 64Cu to determine the formation of metal-phosphate complexes were unsuccessful, probably due to competing protons in the acidic environment of this acidophile. Therefore, we could not determine the efflux of the metal from the cells. Until the formation of copper–phosphate complexes is demonstrated in S. metallicus, we cannot rule out that copper may be exerting an indirect effect that alters polyP levels.
It is possible that the proposed model for polyP-mediated metal detoxification also operates in S. acidocaldarius and S. solfataricus. However, their very low levels of polyP synthesized compared to those in S. metallicus make this system less relevant in the former micro-organisms, since they are very sensitive to copper (Dopson et al., 2003), as we have also seen here. The low levels of polyP in S. acidocaldarius and S. solfataricus are not due to lack of phosphate, because in their natural environments, such as Octopus Spring at Yellowstone National Park or the thermal waters from Pozzo Vasca at Vulcano in the Aeolian Islands, southern Italy, phosphate has been found in different ionic forms depending on the pH of the medium (Amend & Shock, 2001).
The proposed model for metal ion detoxification based on the hydrolysis of polyP involves the transport of metal–phosphate complexes out of the cell. It has been proposed that the inorganic phosphate transport (Pit) system could be a possible candidate for this purpose, because it can reversibly transport metal–phosphate complexes (Keasling, 1997; van Veen, 1997). A Pit-like phosphate transport system was searched for in the available genomes of S. solfataricus, Sulfolobus tokodaii and S. acidocaldarius. A Pit-like transporter was not found in these archaea, but instead we found an open reading frame encoding a protein similar to the Pho84 Pi transporter from Saccharomyces cerevisiae. Experimental evidence indicates that yeast Pho84, as Pit does, transports metal–phosphate complexes (Persson et al., 2003). Pho84, like Pit, belongs to the family of Pi : H+ symporters and is a member of the major facilitator superfamily (Pao et al., 1998). The Pho84 transporter is functional only in acidic environmental conditions (Persson et al., 2003). Currently there is no evidence for a Pho84-like transporter in S. metallicus. However, it is interesting that only proteins similar to Pho84 were present in all the available genomes of acidophilic archaea: Sulfolobus tokodaii, S. solfataricus, S. acidocaldarius, Thermoplasma acidophilum, Thermoplasma volcanicum and F. acidarmanus.
The P-type CPX-ATPases are responsible for the transport of heavy metal ions in archaea, bacteria and eukaryotes (Pedone et al., 2004). In S. solfataricus, a putative copper-transporting ATPase, CopA, has been recently expressed, isolated and its catalytic domain crystallized (Deigweiher et al., 2004). CopB and CopZ have been studied recently in F. acidarmanus (Baker-Austin et al., 2005). Although a genomic DNA sequence is not available yet for S. metallicus, our results suggest that this archaeon might have a copper homeostasis similar to that of other micro-organisms; however, no experimental evidence supporting this proposal is available. Irrespective of the possible existence of such metal cation uptake and efflux mechanisms, it is plausible that a polyP-mediated metal tolerance mechanism such as the one recently described for the metal-resistant polyP-accumulating acidophilic bacterium A. ferrooxidans (Alvarez & Jerez, 2004) is also of great functional significance for the survival of an extremophilic biomining archaeon such as S. metallicus.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Aiking, H., Stijnman, A., van Garderen, C., van Heerikhuizen, H. & van't Riet, J. (1984). Inorganic phosphate accumulation and cadmium detoxification in Klebsiella aerogenes CTC 418 growing in continuous culture. Appl Environ Microbiol 47, 374–377.
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Alvarez, S. & Jerez, C. A. (2004). Copper ions stimulate polyphosphate degradation and phosphate efflux in Acidithiobacillus ferrooxidans. Appl Environ Microbiol 70, 5177–5182.
Amend, J. P. & Shock, E. L. (2001). Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria. FEMS Microbol Rev 25, 175–243.[CrossRef]
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Baker-Austin, C., Dopson, M., Wexler, M., Sawers, R. G. & Bond, P. L. (2005). Molecular insight into extreme copper resistance in the extremophilic archaeon Ferroplasma acidarmanus Fer1. Microbiology 151, 2637–2646.
Cardona, S. T., Remonsellez, F., Guiliani, N. & Jerez, C. A. (2001). The glycogen-bound polyphosphate kinase from Sulfolobus acidocaldarius is actually a glycogen synthase. Appl Environ Microbiol 67, 4773–4780.
Cardona, S. T., Chávez, F. P. & Jerez, C. A. (2002). The exopolyphosphatase gene from Sulfolubus solfataricus: characterization of the first gene found to be involved in polyphosphate metabolism in Archaea. Appl Environ Microbiol 68, 4812–4819.
Chávez, F. P., Lünsdorf, H. & Jerez, C. A. (2004). Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate. Appl Environ Microbiol 70, 3064–3072.
Deigweiher, K., Drell, I. V. T. L., Prutsch, A., Scheidig, A. J. & Lübben, M. (2004). Expression, isolation, and crystallization of the catalytic domain of CopB, a putative copper transporting ATPase from the thermoacidophilic archaeon Sulfolobus solfataricus. J Bioenerg Biomemr 36, 151–159.[CrossRef][Medline]
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Received 31 May 2005;
revised 17 October 2005;
accepted 24 October 2005.
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) and polyP levels (
) were determined at the indicated times of growth.
) were added to the assay reaction mixtures.
), 10 mM (
) or 200 mM (
) CuSO4. At the times indicated, the cells were removed by centrifugation and the radioactive Pi released to the medium supernatants was determined. Means±SE (n=4) are plotted.